Naphthylazo inhibition of amyloidosis

ABSTRACT

Amyloid aggregation in animals is inhibited by administering a naphthylazo compound of formula (I), wherein R1 and R2 are hydrogen, alkyl, substituted alkyl, or complete a heterocyclic ring, R3 is hydrogen or alkyl, R4, R5, R6, and R7 are substitutent groups. The compounds are especially useful in preventing and treating Alzheimer&#39;s disease.

This application claims benefit of provisional application Se. No.60/006,230 filed Nov. 2, 1995.

FIELD OF THE INVENTION

This invention concerns a method for inhibiting amyloidosis utilizingnaphthylazo compounds. The invention is a method for diagnosing andtreating diseases characterized by amyloidosis.

BACKGROUND OF THE INVENTION

Amyloid plaque formation is found in a number of diseases, includingAlzheimer's disease, scrapie, bovine spongiform encephalophy,Gerstmann-Straussler Syndrome, and the like. The amyloid plaquescomprise proteins bound together in a fibrinous matrix. Amyloidosis isthe general name given to diseases and conditions characterized by thepresence of amyloid protein. A number of different types of amyloidprotein are known, and all types are considered pathological, since nonormally occurring amyloids are known. Accordingly, the presence ofamyloid protein in a host is an indication of abnormal formation offibrils and plaques. Amyloidosis has been clinically observed in anumber of disease states, including certain mental illnesses,neurological diseases, and collagenosis. Indeed, the brains of subjectsdiagnosed with Alzheimer's disease have one thing in common, namely anabundance of amyloid in the form of plaques and tangles.

Alzheimer's disease is a degenerative brain disorder characterizedclinically by progressive loss of memory, cognition, reasoning,judgement, and emotional stability that gradually leads to mentaldeterioration and ultimately death. To date, only one clinicallyapproved treatment is available, namely tacrine hydrochloride (Cognex®,from the Parke-Davis Division of Warner-Lambert Company). BecauseAlzheimer's disease and related degenerative brain disorders are a majormedical issue for an aging population, the need for new treatments andmethods for diagnosing the disorders are needed.

WO 9401116 describes the use of congo red, a biphenyl naphthylazo dye,to treat amyloidogenic diseases. We have now discovered that certainnaphthylazo compounds inhibit amyloid aggregation for better than congored. The naphthylazo compounds to be utilized in this invention aredescribed as antiparasitic agents by Elslager, et al., in U.S. Pat. No.3,218,309, which is incorporated herein by reference for its teaching ofsynthesis.

SUMMARY OF THE INVENTION

This invention provides a method for inhibiting amyloid aggregation in amammal by administering a naphthylazo compound. More particularly, theinvention is a method for preventing amyloidosis comprisingadministering to a mammal an effective amount of a compound having theformula ##STR1## wherein: R¹ and R² independently are hydrogen, C₁ -C₄alkyl, hydroxy C₁ -C₄ alkyl, C₁ -C₄ alkoxy-C₂ -C₄ alkyl, C₃ -C₆cycloalkyl, or R¹ and R² taken together with the nitrogen to which theyare attached complete a heterocyclic ring having from 4 to 12 carbonatoms;

n is 1, 2, or 3;

R³ is hydrogen or C₁ -C₄ alkyl;

R⁴ and R⁵ independently are hydrogen, hydroxy, halo, C₁ -C₄ alkyl, C₁-C₄ alkoxy, phenoxy, benzyloxy, hydroxy-C₁ -C₄ alkyl, hydroxy-C₁ -C₄alkoxy, NR¹ R², SO₂ NR¹ R², R¹ R² N--CH₂ (CH₂)_(n) --(O or NH)_(m) whereR¹, R², and n are as defined above, and m is 0 or 1, and R⁴ and R⁵ whenattached to adjacent carbon atoms can be ##STR2## R⁶ and R⁷independently are hydrogen, hydroxy, halo, C₁ -C₄ alkyl, C₁ -C₄ alkoxy,or R¹ R² N-CH₂ (CH₂)_(n) --(O or NH)_(m) --where R¹, R², m, and n are asdefined above; and the pharmaceutically acceptable acid addition saltsthereof.

In a preferred embodiment, the amyloid aggregation inhibitors utilizedhave the formula ##STR3## where R¹ and R² independently are C₁ -C₄ alkyland R⁴ is hydrogen, halo, SO₂ NH₂, benzyloxy, hydroxy C₁ -C₄ alkyl, NR¹R², or R¹ R² N--CH₂ (CH₂)_(n) --(O or NH)_(m) --, and R⁴ is hydrogen orC₁ -C₄ alkoxy.

In a further preferred embodiment, the amyloid aggregation inhibitorsutilized have the formula ##STR4## where R⁴ is hydrogen, 3-chloro,4-fluoro, benzyloxy, 2-(N,N-diethylamino)ethoxy, 2-hydroxyethoxy,1-hydroxyethyl, aminosulfonyl, dimethylamino, or2-(N,N-diethylamino)ethylamino.

DETAILED DESCRIPTION OF THE INVENTION

In the above formulas, "C₁ -C₄ alkyl" means a straight or branchedcarbon chain such as methyl, ethyl, n-propyl, isobutyl, tert.-butyl, andthe like. The alkyl group can be substituted with hydroxy, for example,1-hydroxyethyl, 2-hydroxypropyl, 4-hydroxybutyl, "C₁ -C₄ alkoxy-C₂ -C₄alkyl" are the foregoing C₂ -C₄ alkyl groups having a similar alkylsubstituent bonded through oxygen. Typical of such groups are2-isopropoxyethyl, 3-methoxybutyl, and the like. The term "C₃ -C₆cycloalkyl" includes groups such as cyclopropyl, cyclobutyl, cyclohexyl,and the like.

In the above formula, R¹ and R² can be taken with the nitrogen to whichthey are attached to form a heterocyclic ring such as pyrrolidine,piperidine, morpholine, homopiperazine, and the like.

"Halo" in the above formulas includes fluoro, chloro, bromo, and iodo.Preferred halo groups are fluoro and chloro. R⁶ and R⁷ in the aboveformulas are substituents in either of the rings.

The compounds to be utilized as amyloid aggregation inhibitors areprepared as described in U.S. Pat. No. 3,218,309. For example, adiazonium compound of the formula ##STR5## is reacted with analpha-naphthylamine of the formula ##STR6##

The naphthylazo compounds readily form salts with any of a number ofcommon acids, for instance mineral acids such as hydrochloric acid,sulfuric acid, phosphoric acid, and organic acids such as oxalic,lactic, tartaric, malonic, and related acids.

The ability of the naphthylazo compounds of the above formulas toinhibit amyloid aggregation has been established in a standard in vitroassay. The assay is carried out by mixing beta amyloid peptide (1-40)with radioiodinated (I¹²⁵) labeled peptide to a concentration of 2.5mg/mL in hexafluoroisopropanol. The mixure is diluted 1 to 5 with water(v/v). Ten milliliters of the solution is mixed with 25 μL of 25 mMsodium phosphate buffer pH 6.0. The mixture is allowed to aggregate for2 hours at room temperature with and without a test compound present.The mixtures are then diluted to 235 μL with phosphate buffer to stopthe aggregation process. The solutions are passed through a 0.2-μmmillipore filtermat. Aggregated protein remains in the filter well. Thefilter plate is washed with 50 μL of phosphate buffer and then soaked insolid gel scintillant and counted on a Microbeta counter to determinethe amount of aggregation in the presence of a test compound versuscontrol with no test compound.

Several representative naphthylazo compounds have been evaluated andshown to inhibit amyloid aggregation. The following table presents theactivity of selected compounds, reported as the molar concentration ofcompounds required to cause a 50% inhibition (IC₅₀) of amyloidaggregation in the above assay. Congo red exhibited an IC₅₀ of greaterthan 1000 μM in the test.

                                      TABLE I    __________________________________________________________________________    1 #STR7##                                               IC.sub.50    R.sup.4          R.sup.5   R.sup.6                                   R.sup.7     (μM)    __________________________________________________________________________    H                Cl        H   H           17.8    H                H         H   H           13.7    benzyloxy        H         H   H           15.8    --SO.sub.2 NH.sub.2                     H         H   H           7.0    2 #STR8##        H         H   H           10.5    3 #STR9##        H         H   H           16.1    --OCH.sub.2 CH.sub.2 OH                     H         H   H           18.6    H                H         H                                   7 #STR10##  9.9    4 #STR11##       H         H   H           8.4    5 #STR12##       H         H   H           14.4    F                H         H   H           34    H                H         H   OCH.sub.3   39    H                H         OCH.sub.3                                   H           17                     6 #STR13##                               H   H           30    __________________________________________________________________________

For inhibition of amyloid aggregation according to this invention, allthat is required is to administer to a mammal an effective amount of anaphthylazo compound as defined above. An "effective amount" as usedherein is that quantity of naphthylazo compound which inhibitsaggregation of amyloid protein without causing unacceptable toxiceffects. Typical doses which are effective will be from about 0.1 toabout 1000 mg/day, and more typically from about 50 to about 500 mg/day.The compounds can be administered from one to about three times a dayfor either prophylactic or therapeutic treatment of diseases related tothe deposition of one or more amyloidogenic proteins, for exampleAlzheimer's disease, Down's syndrome, and in general advanced aging ofthe brain.

The naphthylazo compounds can be formulated for convenientadministration orally or parenterally, for instance by intravenous orintramuscular routes. The compounds also are well suited to transdermaldelivery, and can thus be formulated as patches, creams, lotions, andthe like. Typical formulations for oral administration will be made bymixing the naphthylazo compound with common diluents and excipients suchas corn starch, sugar, talc, and the like, and forming tablets,capsules, caplets, syrups, suspensions, and the like. For parenteraldelivery, the compounds are ideally dissolved in isotonic saline oraqueous glucose for injection or intravenous delivery. The compounds canalso be formulated with waxes and polymers and molded into suppositoriesor other common sustained-release delivery forms. The naphthylazocompounds are preferably converted to pharmaceutically acceptable saltsto increase solubility and facilitate formulation and administration.

Because the naphthylazo compounds described above are also effective atbinding to amyloids, they can additionally be utilized to detect amyloiddeposition, and thus to detect disease states associated with amyloidaggregation, such as Alzheimer's disease.

The compounds can readily be radiolabeled with common radioisotopes suchas I¹²⁵, tritium, or the like. For example, compounds wherein R⁴ or R⁵are halo can be made with I¹²⁵. The radiolabeled compounds aresynthesized employing common synthetic techniques utilizing readilyavailable radioactive chemicals. The radiolabeled compound is thenformulated and administered to a mammal in the same manner as describedabove for nonradiolabeled compounds. The mammal can then be scanned withcommon imaging sensors and equipment to detect amyloid deposition andaggregation.

We claim:
 1. A method for inhibiting amyloid aggregation in a mammalcomprising administering an effective amount of a compound having theformula ##STR14## wherein: R¹ and R² independently are hydrogen, C₁ -C₄alkyl, hydroxy C₁ -C₄ alkyl, C₁ -C₄ alkoxy-C₂ -C₄ alkyl, C₃ -C₆cycloalkyl, or R¹ and R² taken together with the nitrogen to which theyare attached complete a heterocyclic ring having from 4 to 12 carbonatoms;n is 1, 2, or 3; R³ is hydrogen or C₁ -C₄ alkyl; R⁴ and R⁵independently are hydrogen, hydroxy, halo, C₁ -C₄ alkyl, C₁ -C₄ alkoxy,phenoxy, benzyloxy, hydroxy-C₁ -C₄ alkyl, hydroxy-C₁ -C₄ alkoxy, NR¹ R²,SO₂ NR¹ R², R¹ R² N--CH₂ (CH₂)_(n) --(O or NH)_(m) where R¹, R², and nare as defined above, and m is 0 or 1, and R⁴ and R⁵ when attached toadjacent carbon atoms can be ##STR15## R⁶ and R⁷ independently arehydrogen, hydroxy, halo, C₁ -C₄ alkyl, C₁ -C₄ alkoxy, or R¹ R² N--CH₂(CH₂)_(n) --(O or NH)_(m) --where R¹, R², m, and n are as defined above;and the pharmaceutically acceptable acid addition salts thereof.
 2. Themethod according to claim 1 employing a compound having the formula##STR16## where R¹ and R² independently are C₁ -C₄ alkyl and R⁴ ishydrogen, halo, SO₂ NH₂, benzyloxy, hydroxy C₁ -C₄ alkyl, NR¹ R², or R¹R² N--CH₂ (CH₂)_(n) --(O or NH)_(m) --, and R⁶ is hydrogen or C₁ -C₄alkoxy.
 3. The method according to claim 2 employing a compound whereinR⁶ is selected from C₁ -C₄ alkoxy and --OCH₂ CH₂ N(CH₂ CH₃)₂.
 4. Themethod according to claim 3 employing a compound selectedfrom:N,N-diethyl-N'-4-(phenylazo)-6-(2-N,N-diethylaminoethoxy)-1-naphthyl!-ethylenediamine;N,N-diethyl-N'- 4-(phenylazo)-6-methoxy-1-naphthyl!ethylenediamine; andN,N-diethyl-N'- 4-(phenylazo)-7-methoxy-1-naphthyl!ethylenediamine. 5.The method according to claim 1 employing a compound having the formula##STR17## where R⁴ is hydrogen, 3-chloro, 4-fluoro, benzyloxy,2-(N,N-diethylamino)ethoxy, 2-hydroxyethoxy, 1-hydroxyethyl,aminosulfonyl, dimethylamino, or 2-(N,N-diethylamino)ethyl-amino.
 6. Themethod according to claim 5 employing a compound selectedfrom:N,N-diethyl-N'- 4-(3-chlorophenylazo)-1-naphthyl!ethylenediamine;N,N-diethyl-N'- 4-(4-fluorophenylazo)-1-naphthyl!ethylenediamine;N,N-diethyl-N'- 4-(phenylazo)-1-naphthyl!-ethylenediamine;N,N-diethyl-N'- 4-(4-benzyloxyphenylazo)-1-naphthyl!ethylenediamine;N,N-diethyl-N'- 4-(4-aminosulfonylphenylazo)-1-naphthyl!ethylenediamine;N,N-diethyl-N'-4-(4-(2-N,N-diethylamino-ethoxy)phenylazo)-l-naphthyl!ethylenediamine;N,N-diethyl-N'-4-(4-(2-hydroxyethoxy)-phenylazo)-1-naphthyl!ethylenediamine;N,N-diethyl-N'-4-(4-(2-N,N-diethylamino-ethylamine)phenylazo)-1-naphthyl!ethylenediamine;N,N-diethyl-N'-4-(4-dimethylamino)-phenylazo)-1-naphthyl!ethylenediamine; andN,N-diethyl-N'-4-(3-(1-hydroxyethyl)-phenylazo)-1-naphthyl!ethylenediamine
 7. Themethod according to claim 1 wherein the amyloid aggregation occurs in amammal having Alzheimer's disease.
 8. A method of diagnosing a mammalhaving amyloid aggregation comprising administering an effective amountof a radiolabeled compound of the formula ##STR18## wherein: R¹ and R²independently are hydrogen, C₁ -C₄ alkyl, hydroxy C₁ -C₄ alkyl, C₁ -C₄alkoxy-C₂ -C₄ alkyl, C₃ -C₆ cycloalkyl, or R¹ and R² taken together withthe nitrogen to which they are attached complete a heterocyclic ringhaving from 4 to 12 carbon atoms;n is 1, 2, or 3; R³ is hydrogen or C₁-C₄ alkyl; R⁴ and R⁵ independently are hydrogen, hydroxy, halo, C₁ -C₄alkyl, C₁ -C₄ alkoxy, phenoxy, benzyloxy, hydroxy-C₁ -C₄ alkyl,hydroxy-C₁ -C₄ alkoxy, NR¹ R², SO₂ NR¹ R², R¹ R² N--CH₂ (CH₂)_(n) --(Oor NH)_(m) where R¹, R², and n are as defined above, and m is 0 or 1,and R⁴ and R⁵ when attached to adjacent carbon atoms can be ##STR19## R⁶and R⁷ independently are hydrogen, hydroxy, halo, C₁ -C₄ alkyl, C₁ -C₄alkoxy, or R¹ R² N--CH₂ (CH₂)_(n) --(O or NH)_(m) --where R¹, R², m, andn are as defined above; and wherein at least one atom is radioactive,and the pharmaceutically acceptable acid addition salts thereof, andimaging the mammal to determine the accumulation of the compound inbrain tissue.